The objective of this research is to continue the development of an expanded molecular encoding system designed by strict analogy with DNA and RNA, but incorporating non-standard bases (NSBs), heterocycles that can form Watson-Crick pairs structurally similar to those formed by natural bases, but joined by non-standard hydrogen bonding patterns. This grant will allow research, in progress at the Swiss Federal Institute of Technology in Zurich, to continue when this laboratory moves to the University of Florida. We have shown that nucleosides bearing non-standard bases form independently replicatable units that increase the number of replicatable" letters" in the DNA and RNA "alphabets", and can expand the genetic "lexicon" to permit messenger RNA to encode more than 20 amino acid "words". We will (a) synthesize NSB-bearing nucleosides and nucleotides, (b) optimize the structures of NSBs to make t hem best suited to be components of an expanded molecular encoding system, (c) characterize the acid-base properties, tautomeric equilibria, conformation in solution binding, and other physical properties of NSBs, (d) continue our work screening polymerase collections to uncover new polymerases that accept NSBs, (e) explore the ability of klenow fragment of DNA pol I, HIV-1 reverse transcriptase, mammalian polymerases alpha, beta, and epsilon, the thermostable DNA polymerases from the archaebacterial thermophile: Pyrodictium abyssi and Pyrodictium occultum, the thermostable DNA polymerase from the eubacterial thermophile Thermotoga maritima, Vent polymerase, AMV reverse transcriptase, T7 RNA polymerase, and other polymerases that may be uncovered in our screening efforts, to accept each of the NSBs, (f) develop polymerase chain reaction technology that will allow the amplification of oligonucleotides containing NSBs (g) expand the scope on in vitro evolution (Selex) experiments based on the expanded set of nucleosides, (h) continue our work using NSBs as tools to encode non- standard amino acids into polypeptides via ribosome-based translation of mRNA molecules, (i) study mutant forms of the Klenow fragment, HIV-1 reverse transcriptase, mammalian pol beta, and the DNA polymerase from T. maritima to accept NSBs, (j) prepare NSBs bearing side chains that carry additional side chain functionality, and (k) develop inhibitors of thrombin using Selex experiments and an expanded genetic alphabet. Collaborations will involve Prof. Ulrich Hubscher at the University of Zurich and professor Catherine Joyce at Yale University. Further, by continuing to do basic research in an area central to disease and disease process and by placing a novel class of molecules in interaction with biological systems, our work will continue to yield unexpected new discoveries relevant to diseases and disease processes.